Dual inhibition of BRD4 and MDM2 cooperate to eradicate AML

Latif, Anne-Louise (2017) Dual inhibition of BRD4 and MDM2 cooperate to eradicate AML. PhD thesis, University of Glasgow.

Abstract

There is a crucial requirement for novel therapies for Acute Myeloid Leukemia (AML). Bromodomain and extra-terminal domain (BET) inhibitors are emerging as active therapeutic agents for hematopoietic malignancies. Pharmacological inhibition of BET bromodomains targets malignant cells by preventing reading of acetylated lysine residues, thus disrupting chromatin-mediated signal transduction, which reduces transcription at oncogenic loci. However, although early phase clinical trials have shown BET inhibitors possess single agent activity against AML (Dombret et al., 2014), complete remission (CR) rates are low and effective combination partners are therefore needed to fully exploit the therapeutic potential of these non-genotoxic agents.
The first aim of this thesis was to demonstrate single agent activity of BET inhibition against a panel of human AML cell lines, followed by testing if cell death with single agent BET inhibition could be enhanced by treatment with the addition of the standard therapies cytarabine (AraC) or azacytidine. We found BET inhibition to be toxic in a dose dependent manner against human AML cell lines, and cell death was enhanced by the addition of AraC. However, this was only found to achieve a synergistic combination index at a one fixed dose ratio in one AML cell line, with additive effects seen in others.
Although a heterogeneous disease, most AML retains wild type TP53. However, TP53 is often rendered functionally deficient by over-expression of MDM2. Restoring the TP53 response though MDM2 antagonism is therefore potentially beneficial to most AML subtypes. We therefore hypothesized that dual inhibition of MDM2 and BET would be synthetic lethal to TP53 wild type AML. In comparison with the combination of BET inhibition with AraC, we found that the combination of the MDM2 antagonist nutlin-3 with a BET-inhibitor (CPI203, Constellation Pharmaceuticals), was synergistically toxic against a variety of TP53 wild type human AML cell lines across several fixed dose ratios, and the combination superior to the single agents when tested on primary murine AML samples.
Given this combination of BET inhibition with MDM2 antagonism proved the most effective combination strategy in terms of cell kill, we then sought to asses the molecular basis of the combination’s synergy. Firstly we asked if the drugs were synergising at the level of their respective targets, i.e. if the BET inhibitor CPI203 in combination with nutlin-3 stabilises TP53 more effectively than single agent nutlin-3 or if the drug combination abrogates MYC more than single agent CPI203. This was not the case, and so we then investigated if the drugs were synergising at the level of the target genes of these key hubs, and through an RNA-seq analysis demonstrated that TP53 target genes were up-regulated by the drug combination.
Given the synergy obtained against several types of AML in vitro on human cell lines with these two non-genotoxic agents, we were keen to test if this could be replicated in vivo. To evaluate the combination in vivo, we used a Trib-2 driven primary AML where leukemogenesis is induced through inhibition of C/EBPα. Treatment was commenced in all mice (n=40), post confirmation of disease engraftment. Three mice from each treatment group were sacrificed after 48hrs and cells sorted for GFP to perform RNA seq in this in vivo setting. After 21 days of treatment all mice were sacrificed (n= 27, one vehicle control succumbed to disease 15 days post engraftment). End of treatment results demonstrated superior in vivo efficacy of dual inhibition of MDM2 and BET in comparison with controls in eradicating AML, p<0.0001. Importantly, normal haematopoiesis was spared - as evidenced by normal full blood counts and comparable myeloid, B-cell and T-cell populations with our C57bl6 wild type controls. RNA-seq of the murine blasts revealed that many more genes significantly (FDR<0.05) changed expression in the combination treated mice than single agent treated mice. The TP53 pathway was the most common up-stream regulator of genes changing expression post combination treatment, p<0.0001. The combination affected many more genes in the p53 pathway than RG7112 alone (120 genes versus 20 genes respectively), in line with our in vitro results.
Ultimately our conclusion is that this combination of BET and MDM2 inhibition is effective and superior to single agent therapy on all TP53 wild type AMLs tested, both in vitro and in vivo. In both contexts this is associated with potentiating the TP53 response and this could be relevant to many patients with TP53 wild type AML. Work on this combination is on-going and we are aiming to take this novel combination forward to a clinical trial in 2017.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: AML, BET inhibitor, MDM2 antagonist.
• Subjects: Q Science > Q Science (General); R Medicine > R Medicine (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Adams, Professor Peter
• Date of Award: 2017
• Embargo Date: 17 May 2021
• Depositing User: Dr Anne-Louise Latif
• Unique ID: glathesis:2017-8189
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 May 2017 08:01
• Last Modified: 17 May 2020 10:25
• URI: https://theses.gla.ac.uk/id/eprint/8189